PROJECT 2 SUMMARY The collecting duct (CD) endothelin-1 (ET-1) system plays a key role in regulating blood pressure (BP) and urinary Na+ excretion. ET-1 directly inhibits CD Na+ and water reabsorption; CD-specific knockout of ET-1 causes salt-sensitive hypertension. ET receptor antagonist-induced fluid retention, a major factor limiting the success of these agents in clinical trials, is largely due to blockade of CD ET-1 actions. Given that CD ET-1 modulates body fluid volume (BFV) homeostasis, it is important to define how this system is regulated by changes in BFV status. Urinary ET-1 excretion is increased in response to salt or water loading; since urinary ET-1 largely derives from the CD, these findings suggest that BFV regulates CD ET-1 production. The mechanism(s) by which BFV expansion enhances CD ET-1 synthesis are incompletely understood. We have found that flow enhances ET-1 production by the inner medullary CD (IMCD) through activation of Ca2+- depending signaling and may require primary cilia and polycystins-1 and -2; these findings raise exciting possibilities about the role of primary cilia and polycystins in the regulation of CD ET-1 and CD salt and water reabsorption. In addition, we found that the purinergic receptors P2Y2 and P2X7 may be important in the IMCD ET-1 flow response, suggesting a link between acutely (ATP) and chronically (ET-1) acting natriuretic and diuretic factors in the CD. We have also found that nitric oxide (NO), likely via NO synthase 3, is necessary for flow stimulation of IMCD ET-1; since NO has never been shown to increase ET-1, this represents a novel and important area of investigation. Taken together, these new findings form the basis for our key hypotheses: 1) Flow increases IMCD ET-1 production through an interplay between cilia, polycystins-1 and -2, P2Y2 and P2X7 receptors, NO and Ca2+-depending signaling; and 2) Cilia, polycystins-1 and -2, P2Y2 and P2X7 receptors, and NO modulation of CD ET-1 affects CD salt and water handling and BP. The following specific aims will be addressed: 1) Evaluate cilia and polycystin regulation of CD ET-1 synthesis and renal function. This involves determining if cilia and polycystins-1 and -2 in the CD regulate BP and salt and water excretion under physiological conditions, if ET-1 plays a role in such regulation, and how cilia and polycystins-1 and -2 in the CD modulate ET-1 production. 2) Evaluate purinergic regulation of CD ET-1 synthesis and renal function. This involves determining if P2Y2 and P2X7 modulate BP and urinary salt and water excretion under physiological conditions, if CD ET-1 plays a role in such regulation, and how P2Y2 and P2X7 in the CD modulate ET-1 production. 3) Evaluate NO regulation CD ET-1 synthesis and renal function. This involves identification of the role of specific NOS isoforms in the CD in modulating BP and urinary salt and water excretion under physiological conditions, if CD ET-1 plays a role in such regulation, and how NOS isoforms in the CD modulate ET-1 production. Taken together, this studies will greatly enhance understanding of how tubule fluid flow regulates CD salt and water reabsorption and the role of ET-1 in this process.